Proliferation of hepatic stem cell progenies (often referred to as oval cells) is responsible for liver regeneration when the replicative and functional capacity of hepatocytes is impaired. The signaling pathways that control stem cell activation remain poorly understood. We investigated the involvement of NF-kB and STAT3 in oval cell-mediated liver regeneration. Oval cells were induced by the AAF/PH protocol. A surface marker of early oval cells, OV1, was used for isolation of pure sub-populations of oval cells either by FACS or MACS. Gene expression profiles and proliferative status of isolated oval cells were examined by Real Time PCR, microarray and FACS. Activation of NF-kB and STAT3 was analyzed by EMSA and Western Blotting. Based on intensity of OV1 staining, we defined three distinct sub-populations of oval cells as OV1low ,OV1medium, and OV1high and showed that they represent different stages of differentiation along hepatocyte lineage. OV1low cells had the properties closest to hepatic stem cells as judged by high expression of c-kit and lack of hepatocyte differentiation markers whereas OV1high cells lost c-kit expression, acquired more mature phenotype and were the most proliferative. Notably, NF-kB was uniformly activated in all three sub-populations of oval cells. In contrast, phosphorylation of STAT3 was only detected in OV1high cells. We conclude that transcriptional activity supported by NF-kB and STAT3 is required for oval cell activation, expansion and differentiation. The differential induction of NF-kB and STAT3 suggest a distinct role for each of these transcription factors at different stages of hepatic stem cell differentiation.